Process for the preparation of 6-hydroxymethyl-2-(β-aminoethylthio)-1-carbadethiapen-2-em-3-carboxylic acid

ABSTRACT

Disclosed is a process for preparing the antibiotic 6-hydroxymethyl-2-(β-aminoethylthio)-1-carbadethiapen-2-em-3-carboxylic acid (I) ##STR1## and its pharmaceutically acceptable salt and ester derivatives. An intermediate for the production of these compounds are compounds of the formula ##STR2## wherein R 5  and R are readily removable protecting groups.

This is a division of application Ser. No. 63,490, filed Aug. 8, 1979,now U.S. Pat. No. 4,252,722 which is a continuation-in-part of U.S. Ser.No. 953,819 filed Oct. 23, 1978, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the total synthesis of the antibiotic6-hydroxymethyl-2-(β-aminoethylthio)-1-carbadethiapen-2-em-3-carboxylicacid (I) and pharmaceutically acceptable salts and esters thereof, whichare disclosed and claimed in co-pending, commonly assigned U.S. patentapplication Ser. No. 933,681 filed Aug. 17, 1978, now abandoned, whichapplication is incorporated herein by reference to the extent that itdiscloses the utility of I as an antibiotic in animal and human therapyand in inanimate systems. ##STR3##

This invention also relates to certain intermediates which are useful inthe synthesis of I.

DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention may conveniently be summarized bythe following reaction diagram: ##STR4## In words relative to the abovediagram, the glycine ester 2 is prepared by reacting X-substitutedacetate 1 with ammonia. Relative to these intermediates species 1 and 2,R¹ is any readily removable carboxyl blocking group such as t-butyl,triphenylmethyl, 2,4-dimethoxybenzyl or the like; and X is a leavinggroup such as chloro, bromo, iodo, or the like. Typically the reaction1→2 is conducted by introducing liquid ammonia to 1 in a sealed vesselat a temperature of from -30° C. to 100° C. for from 1 to 24 hours toprovide 2. Equivalently, the reaction may be conducted at atmosphericpressure at -33° C. (i.e., refluxing solution of liquid ammonia) for1-24 hours.

The reaction 2→3 is accomplished by treating 2 with a maleate diester ina solvent such as ethylacetate, aromatic solvents such as toluene,halogenated alkyls such as CH₂ Cl₂, ether, or the like at a temperatureof from 0° C. to 120° C. for from 1/2 to 24 hours. The ester moieties,R², which define the maleate diester may be selected from any convenientcarboxyl blocking group such as methyl, ethyl, benzyl, or the like.

The aspartic acid intermediate 3 is N-protected according to thereaction 3→4. R³ is any convenient N-protecting group such ascarbobenzyloxy, carbo-t-butyloxy, carbomethoxy, or the like; andestablishment of R³ is accomplished by reacting the correspondingchloroformate or the like with 3 in an aqueous solvent system at a pH offrom 8 to 14 at a temperature of from 0° C. to 100° C. for from 1/2 to10 hours. Equivalently a nonaqueous system may be used, e.g., CH₂ Cl₂,ether, toluene, EtOAc, or the like with, in either class of solvent,from 1 to 10 molar excess of added base (e.g., trialkylamines, NaHCO₃,Na₂ CO₃, NaOH, or the like) to trap the HCl generated during thereaction. Suitable reagents for the establishment of R³ are:benzylchloroformate, methylchloroformate, di-t-butyldicarbonate and thelike.

The cyclization of 4 to form pyrrolidinone 5 is accomplished by treating4 in a solvent such as THF diethyl ether, 1,2-dimethoxyethane, methanol,or the like with a strong base such as sodium methoxide, sodium hydride,or the like at a temperature of from -60° C. to 80° C. for from 1/4 to10 hours.

Thioketal intermediate species 6 is prepared from 5 by treating 5 withR^(2') SH in a solvent such as methylene chloride, toluene, acetic acid,diethylether, EtOAc or the like in the presence of boron trifluorideetherate (BF₃.OEt₂), HBr, trifluoroacetic acid, or the like at atemperature of from 0° C. to 100° C. for from 1/2 to 10 hours. Themercaptan reagent R^(2') SH is such that R^(2') may be alkyl such asmethyl, ethyl, isopropyl, or the like, aralkyl such as benzyl, or arylsuch as phenyl.

The reaction 6.increment.7 is accomplished by treating 6 in the presenceof a base such as triethylamine, sodium bicarbonate, magnesium oxide,sodium carbonate, NaOH, or the like in a solvent such as CH₂ Cl₂,toluene, ethylacetate, diethylether, or the like with esterified malonylhalide wherein X is halogen such as chloro and R⁴ is any convenientcarboxyl blocking group such as ethyl, t-butyl, methyl, isopropyl,benzyl or the like at a temperature of from 0° C. to 100° C. for from1/2 to 10 hours. Alternatively 6 can be treated with an alkyl hydrogenmalonate and a dehydrating agent, such as N,N'-dicyclohexylcarbodiimide.

Cyclization of 7 to yield 8 is accomplished by treating 7 in a solventsuch as methanol, t-butanol, diethylether, 1,2-dimethoxyethane,tetrahydrofuran or the like with a strong base such as potassiumt-butoxide, sodium methoxide, sodium hydride or the like at atemperature of from 0° C. to 100° C. for from 1 to 48 hours.

The reaction 8→9 is accomplished by heating 8 in an aqueous acidsolution (for example 1 to 12 NHCl) at a temperature of from 0° C. to100° C. for from 178 to 24 hours.

The diazotization reaction 9→10 is accomplished by treating 9 in asolvent such as acetonitrile, CH₂ Cl₂, ether, EtOAc, toluene,dimethylformamide or the like at a temperature of from -50° C. to 60° C.with an azide such as p-toluene sulfonyl azide, p-carboxyphenyl sulfonylazide or the like followed by the addition of a base such astriethylamine, 1,4-diazabicyclo[2.2.2]octane, pyridine, or the like forfrom 0.1 to 10 hours.

The carboxyl protecting group R⁵ is established by the reaction 10→11.Typically this is accomplished from the acid chloride of 10, which isobtained by treating 10 in a solvent such as methylene chloride or thelike, preferably in the presence of a catalytic amount ofdimethylformamide, with a chlorinating agent such as oxalyl chloride,thionyl chloride, phosgene or the like for from 1 to 10 hours at atemperature of from 0° C. to 85° C. Reaction of the resulting acidchloride with an alcohol in a solvent such as methylene chloride, ether,ethylacetate, toluene or the like in the presence of a base such astriethylamine, pyridine, N,N-dimethylaniline or the like establishes thedesired protecting group R⁵. Suitable alcohols for this esterificationinclude benzyl alcohol, p-nitrobenzyl, or the like. Alternatively, 10may be converted to a mixed carbonic anhydride, which is then treated asindicated to establish R⁵. Stepwise oxidation of 11 provides 12.Typically the thioketal 11 in a solvent such as methylene chloride,toluene, ethylacetate or the like is treated with a stoichiometricamount of an oxidizing agent such as m-chloroperbenzoic acid peraceticacid, sodium periodate or the like at a temperature of from -50° C. to80° C. for from 1 to 24 hours. The resulting sulfoxide intermediate in asolvent such as acetonitrile CH₂ Cl₂, Et₂ O, EtOAc, toluene or the likeis treated with a 0.2 to 20 fold excess of a strong aqueous acid such asperchloric, sulfuric, hydrochloric or the like at a temperature of from-10° C. to 80° C. for from 0.1 to 5 hours.

The reaction 12→13 is accomplished by treating ketone 12 in an excess ofN-protected aminoethanethiol in the presence of boron trifluorideetherate at a temperature of from 0° C. to 100° C. for from 1 to 120hours. Suitable β-aminoethanethiol reagents include: ##STR5## wherein R⁵is as defined above and is preferably selected from p-nitrobenzyl,benzyl, 2,4-dimethoxybenzyl and the like.

The ring contraction (13→14) is accomplished by treating 13 in thepresence of an equivalent amount of a base such as imidazole, pyridine,triethylamine or the like in a solvent such as methylene chloride,ether, toluene, tetrahydrofuran, ethyl acetate or the like at atemperature of from -100° C. to 60° C. under ultraviolet radiation (250to 400 nm).

The reaction 14→15 is accomplished by treating 14 in the presence of areducing agent such as diborane, borane-methylsulfide complex or thelike in a solvent such as tetrahydrofuran, 1,2-dimethoxyethane, diethylether or the like for from 1/2 to 10 hours at a temperature of from -50°C. to 85° C.

The reaction 15→16 is accomplished by treating the thioketal 15 in asolvent such as methylene chloride, toluene, ethylacetate or the like inthe presence of 1 to 100 mole% water absorbed on the surface of silicagel or alumina (relative to 15) with sulfuryl chloride or the like.

Double bond isomerization 16→17 is accomplished by treating 16 in asolvent such as dimethylsulfoxide, dimethyl formamide, tetrahydrofuran,toluene or the like in the presence of a base such as1,5-diazabicyclo[5.4.0]undec-5-ene, diisopropylamine or the like at atemperature of from -20° C. to 70° C. for from 1/4 to 24 hours.

Final deblocking 17→1 is accomplished by hydrogenolysis in a solventsuch as dioxane, ethanol, tetrahydrofuran, or the like or an aqueousmixture thereof in the presence of a platinum metal catalyst such aspalladium on charcoal, platinum oxide, or the like under an atmosphereof from 1 to 500 psi hydrogen for from 10 to 300 minutes at 0°-25° C.

Referring again to the reaction diagram, intermediate 5 may beaccomplished in a single step according to the following reactionscheme: ##STR6## wherein all symbolism has been previously explained.

According to this scheme, the above-defined maleate diester is reactedwith a suitably N-protected glycinate ester in the presence of a strongbase such as potassium t-butoxide in a solvent such as toluene, diethylether, tetrahydrofuran, 1,2-dimethoxyethane or the like at a temperatureof from -30° C. to 80° C. for from 1/4 to 6 hours; preferably in thepresence of excess t-butylacetate.

Another variation in the above scheme of total synthesis may bedemonstrated at the level of intermediate 13: ##STR7## wherein allsymbolism has been previously explained; intermediate 16 ties in withthe above-detailed scheme of synthesis. In words relative to the abovevariation, species 13 is converted to 13a on treatment with sulfurylchloride and wet silica gel in a solvent such as CH₂ Cl₂, toluene,ethylacetate or the like at a temperature of from -100° C. to 40° C. forfrom 1 to 60 minutes. Ring contraction according to the above-describedprocedure for the transformation 13→14 accomplishes the transformation13a→13a The transformation 13b→16 is accomplished in a procedure exactlyanalogous to the above described transformation 15→16.

Another variation in the above described scheme of total synthesis maybe explained by the following reaction diagram: ##STR8## wherein allsymbolism has been previously explained. According to this scheme,intermediate 12 (as defined above) is ring contracted by irradiationaccording to the procedure detailed for the transformation 13→14 above.The resulting species 12a is converted to 12b according to a procedureexactly analoguous to that described for the transformation 14→15,above. The reaction 12b→12c is accomplished by treating 12b in a solventsuch as methylene chloride, diethyl ether, toluene, dimethyl formamideor the like with p-toluenesulfonic anhydride, methanesulfonic anhydride,p-toluenesulfonyl chloride, methanesulfonyl chloride and a base such astriethylamine, pyridine or the like at -10° C. to 60° C. for 1/2 to 5hours; wherein X is mesyl or tosyl or the like. Establishment of theaminoethylthio side chain is accomplished by treating 12c withN-(carbo-p-nitrobenzyloxy)-2-aminoethanethiol or the like in a solventsuch as dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide inthe presence of 1 to 2 mole equivalents of a base such as triethylamine,pyridine, or the like, at a temperature of from -10° C. to 50° C. forfrom 1/2 to 10 hours. The resulting product 16 is treated as describedabove in the total synthesis.

The compounds of the present invention (I) are valuable antibioticsactive against various gram-positive and gram-negative bacteria andaccordingly find utility in human and veterinary medicine.Representative pathogens which are sensitive to antibiotics I include:Staphyloccus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillussubtilis, Salmonella typhosa, Pseudomonas and Bacterium proteus. Theantibacterials of the invention are not limited to utility asmedicaments; they may be used in all manner of industry, for example:additives to animal feed, preservation of food, disinfectants, and inother industrial systems where control of bacterial growth is desired.For example, they may be employed in aqueous compositions inconcentrations ranging from 0.1 to 100 parts of antibiotic per millionparts of solution in order to destroy and inhibit the growth of harmfulbacteria on medical and dental equipment and as bactericides inindustrial applications, for example in waterbased paints and in thewhite water of paper mills to inhibit the growth of harmful bacteria.

The products of this invention may be used in any of a variety ofpharmaceutical preparations. They may be employed in capsule, powderform, in liquid solution, or in suspension. They may be administered bya variety of means; those of principal interest include: orally,topically or parenterally by injection (intravenously orintramuscularly).

Such tablets and capsules, designed for oral administration, may be inunit dosage form, and may contain conventional excipients, such asbinding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose,sugar, cornstarch, calcium phosphate, sorbitol, or glycine; lubricants,for example, magnesium stearate, talc, polyethylene glycol, silica;disintegrants, for example, potato starch; or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of aqueous or oily suspensions, or solutions, or they may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example, sorbitol,methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose,or carboxymethyl cellulose. Suppositories will contain conventionalsuppository bases, such as cocoa butter or other glycerides.

Compositions for injection, the preferred route of delivery, may beprepared in unit dosage form in ampules, or in multidose containers. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for reconstitution, at the timeof delivery, with a suitable vehicle, such as sterile water.

The compositions may also be prepared in suitable forms for absorptionthrough the mucous membranes of the nose and throat or bronchial tissuesand may conveniently take the form of liquid sprays or inhalants,lozenges, or throat paints. For medication of the eyes or ears, thepreparation may be presented in liquid or semi-solid form. Topicalapplications may be formulated in hydro-phobic or hydrophilic bases asointments, creams, lotions, paints, or powders.

The dosage to be administered depends to a large extent upon thecondition and size of the subject being treated as well as the route andfrequency of administration--the parenteral route by injection beingpreferred for generalized infections. Such matters, however, are left tothe routine discretion of the therapist according to the principles oftreatment well known in the antibiotic art. In general, a daily dosageconsists of from about 5 to about 600 mg of active ingredient per kg. ofbody weight of the subject in one or more treatments per day. Apreferred daily dosage for adult humans lies in the range of from about10 to 240 mg. of active ingredient per kg. of body weight.

The compositions for human delivery per unit dosage, whether liquid orsolid, may contain from 0.1% to 99% of active material, the preferredrange being from about 10-60%. The composition will generally containfrom about 15 mg. to about 1500 mg. of the active ingredient; however,in general, it is preferable to employ a dosage amount in the range offrom about 250 mg. to 1000 mg. In parenteral administration, the unitdosage is usually the pure zwitterionic compound in sterile watersolution or in the form of a soluble powder intended for solution.

In the foregoing word description of the above schematic reactiondiagram for the total synthesis of thienamycin, it is to be understoodthat there is considerable latitude in selection of precise reactionparameters. Suggestion of this latitude and its breadth is generallyindicated by the enumeration of equivalent solvent systems, temperatureranges, protecting groups, and range of identities of involved reagents.Further, it is to be understood that the presentation of the syntheticscheme as comprising distinct steps in a given sequence is more in thenature of a descriptive convenience than as a necessary requirement; forone will recognize that the mechanically dissected scheme represents aunified scheme of synthesis and that certain steps, in actual practice,are capable of being merged, conducted simultaneously, or effected in areverse sequence without materially altering the progress of synthesis.

The following examples recite a precise scheme of total synthesis. It isto be understood that the purpose of this recitation is to furtherillustrate the total synthesis and not to impose any limitation. Alltemperatures are in °C.

EXAMPLE 1 Step A: Preparation of Glycine t-butyl ester ##STR9## Asolution of 150.6 g (1.0 mole) of t-butyl chloroacetate and 300 ml ofliquid ammonia is stirred in an autoclave at room temperature (25° C.)for 3 hours. The solution is vented and concentrated. Ethyl ether (300ml) is added to the residue and filtered. The filtrate is concentratedto give 129.1 g of product as a colorless liquid. Step B: Preparation ofN-(t-butylacetate)aspartic acid dimethyl ester ##STR10## Dimethylmaleate (518 g, 3.92 mole) is added to a solution of 129.1 g (0.98moles) of glycine t-butyl ester in 1.0 liter chloroform at roomtemperature. The mixture is stirred at room temperature for 3 hours andthen extracted with two 300 ml portions of 2 M aqueous hydrochloricacid. The combined aqueous extracts are basified with sodium hydroxideand the product is extracted with ethyl acetate (600 ml). The organicsolution is dried (MgSO₄) and concentrated in vacuo to give 256 g of theproduct as a colorless oil. Step C: Preparation ofN-(t-butylacetate)-N-(carbobenzyloxy)aspartic acid dimethyl ester##STR11## Benzyl chloroformate (165.5 g, 0.97 moles) is added to amixture of 256 g (0.93 moles) of N-(t-butylacetate)aspartic aciddimethyl ester and 84 g (1 mole) of sodium bicarbonate in 500 ml water.The two-phase solution is stirred at room temperature for 3 hours andthen 600 ml EtOAc is added. The organic layer is separated, dried withMgSO₄, and concentrated to an oil. The oil is treated with 1 literhexane, cooled to 0°, and filtered to give the pure product (365 g) ascolorless prisms, m.p. 69°-72°. Step D:1-Carbobenzyloxy-2-carbo-t-butoxy-5-carbomethoxy-3-pyrrolidinone##STR12## To a cold (0°-5°) suspension of 49.7 g (0.92 moles) of sodiummethoxide in 300 ml of tetrahydrofuran (THF) is added a solution of 365g (0.89 moles) of the aspartate derivative in 500 ml of THF over aperiod of 15 minutes. After aging at 0°-5° for 15 minutes the reactionmixture is quenched by addition of 70 g of acetic acid. After removingmost of the solvent in vacuo, the residue is partitioned between EtOAcand two portions of aqueous sodium carbonate solution. The organic layeris dried over MgSO₄ and concentrated to give 225.0 g of crudepyrrolidinone. Step E: Preparation of2-Carboxy-3,3-dithiobenzyl-5-carbomethoxypyrrolidine ##STR13## To anice-cooled solution of 2.0 g. (5.3 mmole) of pyrrolidinone and 2 ml. ofbenzyl mercaptan in 3 ml. of methylene chloride is added 1.5 ml. ofboron trifluoride etherate. The resulting suspension is stirred for 2hours and then 2.0 ml. of trifluoroacetic acid is added and thesuspension warmed to 45° for 3 hours. The yellow solution is cooled toroom temperature and excess saturated aqueous sodium bicarbonate iscautiously added. The white solid is collected by filtration, washedwith several portions of ethyl acetate, and then partitioned between 2 Naqueous hydrochloric acid and ethyl acetate. The organic phase is washedwith water, dried over sodium sulfate and evaporated in vacuo to give0.84 g of a white foamy solid. Step F:2-Carboxy-3,3-dithiobenzyl-5-carbomethoxy-1-(ethoxycarbonylacetyl)pyrrolidine##STR14## To a solution of the amino acid (140.2 g, 0.336 moles) and70.7 g (0.70 moles) of triethylamine in 900 ml CH₂ Cl₂ is added asolution of 54.2 g (0.36 moles) of ethylmalonyl chloride in 100 ml CH₂Cl₂ over a period of 20 min. at room temperature. The resulting mixtureis aged at room temperature for 30 min, then washed with two portions ofwater, dried (MgSO₄), and concentrated in vacuo. The oil is dissolved in800 ml of toluene, cooled to 0° for 3 hrs, and filtered to give 132.5 gof the product as colorless needles. Step G:2-Carboxy-3,3-dithiobenzyl-6,8-dioxo-1-azabicyclo[3.3.0]octane ##STR15##To a solution of the pyrrolidine (151.6 g, 0.29 moles) in 900 ml CH₃ OHat room temperature is added 67.3 g (0.60 moles) of potassium-t-butylalcoholate as a solid over 5 minutes. After aging for 24 hrs, thereaction is quenched by adding 42.1 g (0.70 moles) of acetic acid andthen concentrated in vacuo. To the residue is added 200 ml of 1 M aq.HCl and 100 ml CH₂ Cl₂ and the resulting solid is collected byfiltration to give 144.5 g of the enol ester. The ester is dissolved in1200 ml acetic acid and 600 ml of 2 N aq. HCl and heated to 75°-80° for1 hr. The solution is cooled, diluted with H₂ O, and extracted with twoportions (400 ml each) of ethyl acetate. The combined organic layers aredried (MgSO₄) and concentrated in vacuo to give 105.3 g of the productas a foamy solid. This material is pure enough to use in Step G. Step H:2-Carboxy-3,3-dithiobenzyle-7-diazo-6,8-dioxo-1-azabicyclo[3.3.0]octane##STR16## The bicyclic acid (2.96 g, 6.9 mmole) is dissolved in 50 ml ofacetonitrile, cooled in an ice-bath, and treated first with a solutionof 2.72 g (13.8 mmole) of p-toluenesulfonyl azide in 7 ml acetonitrileand then with a solution of 1.39 g (13.8 mmole) of triethylamine in 7 mlacetonitrile. The resulting brown solution is warmed to room temperatureand aged for 40 minutes, then concentrated in vacuo. The residue isdissolved in ethylacetate and washed successively with 2 N-hydrochloricacid, water, and saturated sodium chloride solution. The organic phaseis concentrated in vacuo to give 6.8 g of a red liquid which ischromatographed on 140 g of silica gel. After a forerun of 1% aceticacid in methylene chloride is taken, the product is eluted with 1%acetic acid in ethyl acetate. Concentration gives 2.94 g of the productas a yellow-brown solid. Step I: ##STR17##2-Carbo-p-nitrobenzyloxy-3,3-dithiobenzyl-7-diazo-6,8-dioxo-1-azabicyclo[3.3.0]octane

To a solution of the acid (2.84 g, 6.27 mmole) in 20 ml methylenechloride is added 2.10 ml of oxalyl chloride followed by 0.10 ml ofN,N-dimethylformamide. The brown solution is aged for 4 hours and thenconcentrated in vacuo to a brown, oily solid. To an ice-cooled solutionof the acid chloride in 16 ml methylene chloride is added a solution of1.06 g (6.9 mmole) of p-nitrobenzyl alcohol in 3 ml methylene chloridefollowed by a solution of 0.76 g (6.27 mmole) of N,N-dimethylaniline in3 ml methylene chloride. The resulting solution is stirred at roomtemperature for 7 hours and then concentrated. The residue is dissolvedin ethylacetate and washed successively with 2 portions of saturatedsodium bicarbonate, H₂ O, 2 portions of 2 N hydrochloride acid andwater. The organic phase is dried over MgSO₄ and concentrated in vacuoto give 3.1 g of crude ester. Purification is effected by chromatographyon 75 g of silica gel. The fractions eluted with 4% ethylacetate inbenzene are concentrated to give 2.45 g of ester as a yellow solid. Theproduct can be further purified by recrystallization from 20 ml ofdiethyl ether to give 2.09 g of yellow prisms, mp=130°-2°.

Step J: ##STR18##2-Carbo-p-nitrobenzyloxy-7-diazo-3,6,8-trioxoazabicyclo[3.3.0]octane

To an ice-cooled solution of the thioketal (1.62, 2.76 mmole) in 20 mlof methylene chloride is added dropwise a solution of 0.48 g (2.76mmole) of m-chloroperbenzoic acid in 10 ml of methylene chloride. Theresulting solution is aged for 15 minutes and then washed with twoportions of saturated sodium bicarbonate and then water. The organicphase is dried of MgSO₄, filtered, and concentrated in vacuo to give thecrude sulfoxide (mixture of isomers) as a yellow solid. To a solution ofthe crude sulfoxide in 2.5 ml of acetonitrile is added 0.53 g of a 72%aqueous solution of perchloric acid diluted with 2 ml of acetonitrile.After stirring for 3 minutes the solution is concentrated in vacuo to ayellow oil. The oil is dissolved in ethylacetate and washed with waterand then two portions of saturated sodium chloride solution. The organicphase is dried over MgSO₄ and concentrated in vacuo to give 1.78 g of ayellow gum. The pure ketone is obtained by crystallization from 50 ml ofethylacetate. The pure product amounts to 0.80 g of yellow prismsm.p.=185 (dec).

Step K: ##STR19##2-Carbo-p-nitrobenzyloxy-3,3-bis(N-carbo-p-nitrobenzyloxy-β-aminoethylthio)-7-diazo-6,8-dioxo-1-azabicyclo[3.3.0]octane

A mixture of the ketone (0.23 g, 0.64 mmole) andN-(carbo-p-nitrobenzyloxy)-β-aminoethanethiol (1.82 g, 7.11 mmole) isdissolved in 0.51 ml of boron trifluoride etherate and 10 ml methylenechloride and aged at room temperature for 3 days. The reaction mixtureis diluted with ethylacetate and washed successively with water, twoportions of 10% lead acetate and solution and water. The organic layeris dried and concentrated in vacuo to give 1.00 g of orange gum.Purification is effected by chromatography on 30 g of silica gel. Thefractions eluted with 40% of ethylacetate in toluene are concentrated togive the pure product as a colorless gum (0.38 g).

Step L: ##STR20##2,2-bis(N-carbo-p-nitrobenzyloxy-β-aminoethylthio)-3-carbo-p-nitrobenzyloxy-1-carbadethiapenam-6-carboxylicacid

A solution of the diazo compound (1.65 g, 1.93 mmole) and imidazole(0.13 g, 1.93 mmole) in 35 ml methylene chloride containing 41 mg (2.30mmole) water is placed in a pyrex vessel fitted with a magnetic stirringbar and a nitrogen inlet tube. The vessel is partially immersed in adry-ice methanol bath and the solution is thoroughly flushed withnitrogen. The solution is then irradiated for 120 minutes from adistance of 11-15 cm with a 450 watt Hanovia high-pressure mercury vaporlamp fitted with a reflector. The solution is warmed to about 0° andcharged on a column of 35 g of silica gel packed in methylene chloride.After a forerun of 25% ethylacetate in benzene, the product is elutedwith a mixture of 1% acetic acid and 30% ethyl acetate in methylenechloride. Concentration in vacuo gives the acid (0.68 g) as apale-yellow gum.

Step M: ##STR21##6-hydroxymethyl-2,2-bis(n-carbo-p-nitrobenzyloxy-β-aminoethylthio)-3-carbo-p-nitrobenzyloxy-1-carbadethiapenam

A 1 M solution (2.69 ml, 2.69 mmole) of borane in tetrahydrofuran isadded dropwise to a solution of the β-lactam acid (1.53 g, 1.81 mmole)in 15 ml of anhydrous 1,2-dimethoxyethane at 0° C. The solution is agedat 0° for 50 minutes and then quenched by addition of 1.5 ml of aceticacid. The solution is diluted with ethylacetate and washed with threeportions of water, dried, and concentrated in vacuo to give the crudealcohol as a nearly colorless gum (1.49 g). Chromatography on 30 g ofsilica gel and concentration of the fractions eluted with 50%ethylacetate in benzene gives the pure alcohol (0.69 g) as a colorlessgum.

Step N: ##STR22##6-hydroxymethyl-2-(N-carbo-p-nitrobenzyloxy-β-aminoethylthio)-3-carbo-p-nitrobenzyloxy-1-carbadethiapen-1-em

To a cold (-60°) suspension of the thioketal (0.58 g, 0.70 mmole) andwet silica gel (0.11 g of silica and 0.11 g of water) in 15 ml ofmethylene chloride is added a solution of sulfuryl chloride (0.11 g,0.84 mmole) in 1 ml of methylene chloride. The suspension is stirred at-60° for 15 minutes then 5 ml of pH 7.5 aq. phosphate buffer andethylacetate is added. The organic layer is washed with water, dried andconcentrated to give the crude vinyl sulfide. Chromatography on 13 g ofsilica gel and elution with 45% ethylacetate in benzene gives the purevinyl sulfide (0.20 g) as a pale yellow gum.

Step O: ##STR23##6-hydroxymethyl-2-(N-carbo-p-nitrobenzyloxy-β-aminoethylthio)-3-carbo-p-nitrobenzyloxy-1-carbadethiapen-2-em

The vinyl sulfide (0.29 g, 0.51 mmole) is dissolved in 5 ml of drydimethyl sulfoxide containing 1,5-diazabicyclo[5.4.0]undec-5-ene (0.074g, 0.49 mmole). The solution is aged at room temperature for 15 minutesand then quenched by addition of 5% aq. potassium dihydrogen phosphate.The product is extracted into ethylacetate, washed with three portionsof water, dried and concentrated to give a yellow gum which ischromatographed on 9 g of silica gel. The 45% ethylacetate in benzenefractions are concentrated to give 0.080 g of recovered startingmaterial. The 70% ethyl acetate in benzene fractions afford the pureproduct (0.081 g) as a yellow gum.

Step P: ##STR24##

A mixture of vinyl sulfide (0.10 g, 0.17 mmole), and 50 mg of 10% Pd/C,and K₂ HPO₄ (40 mg, 0.23 mmole) in dioxane (1 ml), ethanol (1 ml), anddeionized water (7 ml) is pressurized to 50 psi with hydrogen. Themixture is shaken or stirred at room temperature for 50 minutes and thenit is vented and filtered. The catalyst is washed with 2 ml of 0.1 N pH7 phosphate buffer. The combined filtrates are concentrated in vacuo tothe cloud point and then extracted with ethyl acetate. The water layeris concentrated to about 3 ml and charged on a column of 110 g XAD-2resin. The column is eluted with fractions monitored by UV. Thosefractions with UV absorption at 300 mμ are combined and lyophilized togive the product as a white solid (12.3 mg).

EXAMPLE 2 Step A:1-Carbobenzyloxy-2-carbo-t-butoxy-5-carbomethoxy-3-pyrrolidinone##STR25##

To an ice-cooled suspension of 11.2 g. (0.10 mole) of potassiumtert-butyl alcoholate and 125 ml. of dry toluene is rapidly added amixture of 70 g. (0.60 mole) of t-butyl acetate and 26.5 g. (0.10 mole)of N-carbobenzyloxy-t-butylglycinate. The resulting suspension is agedfor 5 minutes and then a solution of 14.4 g. (0.10 mole) of dimethylmaleate is added dropwise. The brown solution is aged at 0° C. for 20minutes and then quenched by rapid addition of 25 ml. of glacial aceticacid. The organic solution is washed successively with water, 2 portionsof solid aqueous sodium carbonate and 2 portions of water, then dried(MgSO₄) and evaporated in vacuo to give 30.6 g. of viscous, yellow oil.

The crude product is purified by reacting it with 19 g. (0.11 mole) ofGirard's Reagent T (carboxymethyl)-trimethylammonium chloride hydrazide)and 12 ml. of glacial acetic acid in 300 ml. of methanol at 55° C. for 2hours. The solution is concentrated in vacuo. The residue is partitionedbetween 60 ml. H₂ O and 60 ml. ethyl acetate. The aqueous layer isseparated, 60 ml. of diethyl ether is added, and 15 ml. of concentratedhydrochloric acid is added with stirring. After 10 min. of stirring, thelayers are separated. The organic phase is washed with water, dried(MgSO₄) and evaporated in vacuo to give 15.8 g. of pure pyrrolidinonewhich slowly solidifies on standing.

Step B: 2-Carboxy-3,3-dithiomethyl-5-carbomethoxy-pyrrolidinehydrobromide ##STR26## The pyrrolidinone (10.0 g, 0.026 mole) isdissolved in 50 ml. of liquid methyl mercaptan. To this refluxingsolution (6°) is added dropwise a solution of 2 g. of hydrogen bromidein 8 ml. of glacial acetic acid. The resulting solution is stirred for12 hours and then concentrated in vacuo. The oily residue is dissolvedin 10 ml. of methanol. With stirring, 400 ml. of ether is added to themethanol solution. The oil which separates is allowed to settle and thesolvent is removed by decantation. This dissolution-precipitationprocedure is repeated once and then the oil is pumped to constant weightto give 10.78 g of foamy, tan solid. Step B¹ :2-Carboxy-3,3-dithiomethyl-5-carbomethoxy-1-(t-butoxycarbonylacetyl)pyrrolidine##STR27## To a stirred suspension of 4.53 g. of amino acid and 2.6 g. ofpotassium t-butyl malonate in 70 ml. methylene chloride is added asolution of 2.70 g. of N,N'-dicyclohexylcarbodiimide. The suspension isstirred for 20 minutes, then cooled to 0° C. and filtered. The filtrateis washed with 1 N aqueous hydrochloric acid and then the product isextracted into saturated aqueous bicarbonate solution. The basic layeris carefully acidified with 2 N hydrochloric acid and the productextracted into 2 portions of methylene chloride. The combined organicextracts are dried over MgSO and concentrated in vacuo to give 1.48 g.of crude prodduct. Purification is affected by chromatography on 40 g.of silica gel. The product which is contained in the fractions elutedwith 1% acetic acid in ethyl acetate amounts to 0.66 g. of colorlessoil. Step C:2-Carboxy-3,3-dithiobenzyl-5-carbomethoxy-1-(t-butoxycarbonylacetyl)pyrrolidine##STR28## To an ice-cooled solution of 4.17 g. (0.01 mole) of amino acidand 1.6 g. (0.01 mole) of t-butyl hydrogen malonate in 70 ml. ofmethylene chloride is added a solution of 2.06 g. (0.01 mole) ofN,N'-dicyclohexylcarbodiimide in 8 ml. of methylene chloride. Theresulting suspension is warmed to room temperature and aged for 2 hoursand then filtered. The filtrate is washed with 2 N hydrochloric acid,dried over MgSO₄, and concentrated in vacuo to an oil. The crude productis chromatographed on 200 g. of silica gel, after a forerun of 1% aceticacid in methylene chloride the product is eluted with 1% acetic acid inethyl acetate. Concentration gives the product (mixture of isomers) as afoamy white solid (3.8 g). Step D:2-Carboxy-3,3-dithiobenzyl-6,8-dioxo-1-azabicyclo[3.3.0]octane ##STR29##To an ice-cooled solution of the pyrrolidine (5.03 g, 9.0 mmole) in 100ml of methanol is added 2.02 g (18.0 mmole) of potassium-t-butylalcoholate. The solution is aged at room temperature for 45 minutes andthen heated to reflux for 30 minutes. The solution is concentrated invacuo. The residue is treated first with 2 N aq. hydrochloric acid andthen extracted with three portions of methylene chloride. The combinedorganic extracts are dried over MgSO₄ and concentrated to an orange gumwhich is dissolved in 100 ml of toluene and refluxed for 2.5 hours.Concentration in vacuo gives 4.2 g of crude product which is purified bychromatography on silica gel (130 g). The product which is eluted with1% acetic acid in ethyl acetate amounts to 3.96 g of foamy, yellowsolid. EXAMPLE 3 ##STR30## Preparation of Pharmaceutical Compositions

One such unit dosage form comprises a blend of 120 mg. of l with 20 mg.of lactose and 5 mg. of magnesium stearate which is placed in a No. 3gelatin capsule. Similarly, by employing more of the active ingredientand less lactose, other dosage forms can be prepared; should it benecessary to mix more than 145 mg. of ingredients together, largercapsules may be employed. Equivalently, compressed tablets and pills canbe prepared. The following examples are further illustrative of thepreparation of pharmaceutical formulations:

    ______________________________________                                        TABLET           PER TABLET                                                   ______________________________________                                        Compound 1 ˜                                                                             125 mg.                                                      Dicalcium Phosphate                                                                            200 mg.                                                      Cornstarch, U.S.P.                                                                              6 mg.                                                       Lactose, U.S.P.  200 mg.                                                      Magnesium Stearate                                                                             270 mg.                                                      ______________________________________                                    

The above ingredients are combined and the mixture is compressed intotablets, approximately 0.5 inch in diameter, each weighing 800 mg.

What is claimed is:
 1. The compound: ##STR31## wherein R⁵ and R arereadily removable protecting groups.